PROJECT SUMMARY (ABSTRACT): Background: N6-methyladenosine (m6A) modification is the most abundant internal modification in eukaryotic messenger RNAs (mRNAs) and plays critical roles in various normal bioprocesses. Evidence is emerging that m6A and the associated machinery also play roles in cancers. Acute myeloid leukemia (AML) is one of the most common and fatal forms of hematopoietic malignancies with distinct cytogenetic (e.g., MLL-rearranged) and molecular (e.g., mutations in FLT3 and NPM1) abnormalities. Over 70% of AML patients cannot be cured by contemporary therapies due to drug resistance and relapse. The leukemia stem/initiating cells (LSCs/LICs) are considered to be the root cause for the treatment failure and relapse of AML. Thus, it is critical to better understand the molecular mechanisms underlying AML pathogenesis, LSC/LIC self-renewal and drug resistance, which may lead to the development of effective novel therapeutic strategies to treat AML. RNA m6A modification is deposited by the METTL3-METTL14 methyltransferase complex (MTC; i.e., writer), and may affect the stability and/or translation efficiency of target mRNAs. We and others have reported that both METTL3 and METTL14 play oncogenic roles in MLL-rearranged AML, but their definitive roles in the development and maintenance of other (i.e., non-MLL- rearranged) subtypes of AMLs and especially in LSC/LIC self-renewal remain elusive. Our unpublished preliminary data imply that METTL3 and METTL14 may also play roles in drug resistance in AML, which warrants further systematic studies. Furthermore, their critical targets in AMLs have yet to be well investigated. Hypothesis: METTL3 and METTL14 play essential oncogenic roles in AML pathogenesis, LSC/LIC maintenance and drug resistance through m6A-dependent regulation of expression of critical target genes. Specific Aims: 1) To determine the roles of METTL3 and METTL14 in the development and maintenance of different subtypes of AMLs, as well as in the self-renewal of relevant LSCs/LICs and normal HSCs; 2) To determine the roles of METTL3 and METTL14 in drug resistance of AMLs; and 3) To decipher the molecular mechanisms underlying the pathological roles of METTL3 and METTL14 in AML development/maintenance, LSC/LIC self-renewal and drug resistance. Study Design: 1) We will use inducible Mettl3 and Mettl14 knockout mouse models coupled with mouse AML models, as well as patient-derived xeno-transplantation (PDX) models, to investigate the roles of METTL3 and METTL14 in development/maintenance of different subtypes of AMLs and self-renewal of LSCs/ LICs and normal hematopoietic stem cells (HSCs). 2) We will conduct both gain- and loss-of-function in vivo studies to determine whether METTL3 and METTL14 play critical roles in drug resistance of AMLs. 3) We will perform PAR- CLIP-seq, m6A-seq, RNA-seq, RNA life-time profiling-seq, and ribosome profiling-seq to identify all direct targets of METTL3 and METTL14 in AMLs and to understand how METTL3 and METTL14 regulate expression of their target RNAs, followed by validation and functional studies of a set of top candidate targets.